This proposal focuses on characterizing mechanisms of HIV-1 spread in vivo after intravenous exposure. Cell-to-cell infection has been shown to be a distinct mechanism of viral spread that is more efficient and rapid in vitro. Currently, the roleof cell-free versus cell-to-cell modes of viral spread has not been examined in vivo. This study hopes to reveal the dominant mode of viral spread that occurs during parenteral transmission, which we hypothesize to be mediated by cell-to-cell contacts rather than cell-free virus. Insight into how HIV-1 propagates in vivo will help to enhance the development of therapeutic agents that can efficiently block all forms of viral spread, ultimately, preventing disease progression of HIV. We will directly visualize cellular interactions of infected CD4+ T cells in live tissue. Usig a humanized mouse HIV-1 infection model, we plan to characterize the initial sites where virus establishes after intravenous HIV exposure to model transmission in intravenous drug users (IDU). For visualization of HIV transmission, the use of fluorescently-tagged HIV-1 clones will be assessed by flow cytometry and novel multi-photon live imaging techniques. The three areas to be investigated in this project are: 1) Characterize organ-to-organ spread of HIV-1 after intravenous injection of cell-associated or cell-free virus. We plan to define the sequential events of organ-to-organ transfer of HIV-1 in humanized mouse models through the use of two-photon live imaging and flow cytometry. In doing so we will elucidate the mechanism of viral trafficking to show the impact lung localization and cellular migration has on virus dissemination. 2) Determine the patterns of co-transmissions to discern between two modes of spread. We aim to utilize a dual infection strategy using distinct fluorescent HIV-1 variants to characterize the function of HIV-1 cell-to-cell spread in cotransmission. To analyze the spread of double infection with different modes of transmission, we will perform two-photon microscopy and flow cytometry on humanized murine tissues. 3) Examine virus transfer through virological synapse in vivo. Synaptic transfer of virus has been readily characterized in vitro; however, the presence of stable synapse formation in vivo has yet to be observed. We aim to visualize the transfer of viral proteins through cell-to-cell contact using time-lapse two-photon intravital imaging of lymphoid organs in humanized mice.